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29 November 2004. Ubiquitination, sumoylation (tagging with small ubiquitin-like modifiers), ubiquitinated aggregates, and problems with ubiquitin-mediated proteasomal degradation have all been linked to pathological processes that underlie various neurodegenerative diseases, including Alzheimer, Parkinson, and Huntington diseases. Researchers studying these processes may be interested in a new technique that can measure ubiquitination—and deubiquitination—in real time. In the December issue of Nature Methods (first published online Nov. 18), Michel Bouvier and colleagues from the University of Montreal, Quebec, describe a way to measure this covalent modification process using bioluminescence resonance energy transfer, commonly known as BRET.

BRET, first described by Carl Johnson and colleagues at Vanderbilt University, Tennessee, is a bit like its cousin FRET (florescence resonance energy transfer—see Alzheimer Research Forum related news story) in that it measures molecular proximity by relying on transfer of light from one molecule to another. But BRET uses a luciferase enzyme as the electron donor, thus eliminating the need to excite samples with light. In their study, Bouvier and colleagues used two hybrid proteins (think of the classical two-hybrid protein-protein interaction assay) in which a bait, in this case a protein fused to Renilla luciferase (RLuc), was used to lure in a prey—ubiquitin fused to green fluorescent protein (GFP).

Joint first authors Julie Perroy and Stephanie Pontier used a β-arrestin fusion as the bait. β-arrestin is a well-known substrate for ubiquitination. When the authors expressed the two hybrids, along with the luciferase substrate DeepBlueC coelenterazine, in human kidney cells, they found that the energy transfer, as judged by GFP fluorescence, dramatically increased as the ubiquitin-GFP concentration was raised. This indicates that the GFP fluorescence was the result of a direct interaction between the arrestin and ubiquitin hybrids.

But this does not necessarily mean that the authors were measuring ubiquitination. They may have just been measuring contact between arrestin and ubiquitin. So to confirm that the BRET signal was actually a reflection of a covalent modification, Perroy and Pontier used a mutant ubiquitin that cannot be covalently attached to substrate protein. Using this prey, no GFP emissions were observed that were above baseline, indicating that for BRET to take place in this system, the ubiquitin hybrid must covalently bind to its substrate.

To show that their detection system is sensitive enough to detect real biological changes, the authors used G-protein coupled receptor (GPCR) activation to induce β-arrestin ubiquitination. When the authors challenged cells with isoproterenol (ISO) or arginine-vasopressin (AVP), agonists for the V2-vasopressin receptor (V2R) and β2 adrenergic receptor (β2AR) GPCRs, respectively, they found significant increases in the BRET signal, indicating that β-arrestin had been ubiquitinated. But because activated β2ARs are known to interact only transiently with arrestin, the authors were able to take this experiment one step further and measure, in real time, the delicate dynamics of the ubiquitination process.

While both GPCR agonists caused a rapid and similarly robust increase in the BRET signal (over fivefold), peaking after about two minutes, the isoproterenol-induced signal persisted for over 10 minutes, whereas the vasopressin-induced signal began to fade as quickly as it peaked, and was all but absent after 10 minutes. “This reduction in BRET signal most likely reflects a deubiquitination process and not a degradation of β-arrestin,” suggest the authors.

One caveat of the method is that to avoid quenching, the GFP-ubiquitin reporter must be designed to prevent polyubiquitination. The authors circumvented this problem by changing ubiquitin lysines 48 and 63, which can bind additional ubiquitin chains, to alanines.

As compared to Western blot analysis, which is typically used to measure ubiquitination of substrates, the BRET assay has several advantages, suggest Bouvier and colleagues: It avoids possible signal alterations that could result from cell lysis; it can capture the dynamic nature of the process; it can be used to monitor changes in specific cells given a specific treatment; and, because of the nature of the BRET measurements, it can distinguish deubiquitination from the simple loss of signal due to degradation (baseline BRET acts as an internal control). It will be of interest to see if this technique could easily be adapted for use in neurons.—Tom Fagan (Alzheimer Research Forum).

Reevaluation of the dopamine D2 receptor in the treatment of schizophrenia: Novel intracellular mechanisms as predictors of antipsychotic efficacy
Since the advent of antipsychotic medications, there have been many speculations about their precise mechanisms of therapeutic action. Although it is apparent that blockade of dopamine D2 receptors (D2R) is crucial to the efficacy of all current antipsychotic medications, it is not clear which signaling events downstream of the D2R must be blocked for the therapeutic actions of antipsychotics and which events, when blocked, lead instead to side effects.

The best characterized D2R-mediated signaling pathways involve coupling of the receptor to pertussis toxin-sensitive G proteins of the Gi and Go subfamilies (Sidhu and Niznik, 2000), through which D2R activation results in a decrease in cyclic AMP (cAMP). D2R activation can also have a number of other effects, including enhancement of specific potassium currents, inhibition of L-type calcium currents, mediation of extracellular signal-regulated kinase 1 (ERK1) and potentiation of arachidonic acid release (Beom et al., 2004; Missale et al., 1998; Perez et al., 2006; Hernández-López et al., 2000). There is growing evidence that D2Rs can interact with a number of membrane-bound or intracellular proteins, which may further modulate signaling specificity (reviewed in Terrillon and Bouvier, 2004; Ferré et al., 2007a). In particular, D2R heteromerization may result in a switch from Gi/o coupling to Gs (i.e., through D2R and cannabinoid 1 receptor interaction) (Kearn et al., 2005) or to coupling with Gq (as suggested in D2R and D1R interactions) (Rashid et al., 2007). Moreover, heteromerization between D2R and other receptors such as the adenosine A2A receptor may allow for reciprocal modulation of D2R function (Ferré et al., 2007a; Ferré et al., 2007b). It also has been suggested that calcium signaling mechanisms may modulate D2R’s signaling efficacy; interaction between D2R and calcium-binding protein S100B results in enhanced D2R intracellular signaling (Liu et al., 2008; Stanwood, 2008).

Using newly developed BRET (bioluminescent resonance energy transfer) biosensors in assays that measure direct protein-protein interactions within the living cell, recent studies have demonstrated that antipsychotic medications prevent arrestin 3 recruitment by blocking D2R activation (Klewe et al., 2008; Masri et al., 2008). Masri et al. (2008) hypothesized that antipsychotic drugs achieve their therapeutic effect through a common mechanism involving blockade of arrestin-mediated signaling (Masri et al., 2008). Masri et al. (2008) also demonstrated that nearly all antipsychotics tested (including haloperidol, clozapine, olanzapine, desmethylclozapine, chlorpromazine, quetiapine, risperidone and ziprasidone) behave as inverse agonists to decrease constitutive G protein signaling as well as to prevent the agonist quinpirole from inhibiting cAMP synthesis (via D2R-mediated Gi/o signaling). The lone exception, aripiprazole, behaved as a partial agonist instead of as an inverse agonist of the G protein mediated effects. The latter finding is consistent with previous studies highlighting aripiprazole’s ability to differentially modulate various G protein-mediated effector pathways, a property termed “functional selectivity” (Mailman, 2007; Urban et al., 2007). Using the BRET assay, Klewe et al. (2008) and more recently Masri et al. (2008) demonstrated that all antipsychotics, including aripiprazole, block arrestin 3 recruitment. This finding has led Masri et al. (2008) to suggest that blockade of arrestin 3 recruitment to the D2R, and not modulation of G-protein-mediated pathways, is a common and specific property of all current antipsychotics and may be used to predict the antipsychotic efficacy of drugs in development (Masri et al., 2008). This hypothesis remains to be tested and at present appears to lean heavily on the evidence for aripiprazole’s atypical effects on constitutive (non-agonist-dependent) D2R-mediated G-protein signaling. Indeed, the fact that lithium acts to prevent arrestin-mediated signaling in response to amphetamine but is not an effective antipsychotic in monotherapy suggests that antipsychotic action may be more complex than simple blockade of D2R-mediated arrestin signaling. In addition, the ability of antipsychotics, including aripiprazole, to block agonist binding to the D2R and thus activation of the receptor, makes it likely that agonist-induced activity in multiple signaling pathways will also be blocked by these drugs.

In a mouse model, acute and chronic haloperidol treatment was shown to increase levels of active, phosphorylated Akt isoform Akt1 and increased phosphorylation and inactivation of GSK-3β (Emamian et al., 2004). Thus, it was suggested that haloperidol treatment may compensate for the decreased levels of endogenous Akt1 in the frontal cortex of people with schizophrenia (Emamian et al., 2004). Atypical antipsychotics also impact on the regulation of Akt and GSK-3β activities. For example, treatment with clozapine results in increased levels of phosphorylated GSK-3β (Kang et al., 2004; Sutton et al., 2007). Interestingly, however, differences between haloperidol and atypical antipsychotics have emerged in the kinetics of Akt/GSK-3 phosphorylation, the levels of proteins expressed following drug exposure, and the signaling pathways that are preferentially activated (Roh et al., 2007).

The abilities of antipsychotic drugs to activate distinct signaling pathways to mediate their ostensible differential pharmacologic effects would suggest clinical variation in their therapeutic effects. However, meaningful differences in the clinical effects of these compounds have not been clearly or consistently evident. The initial reports of superior efficacy of the so-called second generation or atypical antipsychotics on measures of psychosis (Kane et al., 1988), negative symptoms (Tollefson et al., 1997), cognitive deficits (Keefe et al., 1999), relapse prevention (Csernansky et al., 2002), adherence (Wahlbeck et al., 2001) and illness progression (Lieberman et al., 2005a), have not been borne out by more recent studies (Geddes et al., 2000; Lieberman et al., 2005b; Jones et al., 2006; Leucht et al., 2008). Indeed, the differences between antipsychotic drugs are most evident in the types, frequency and severity of side effects rather than in their therapeutic actions (Leucht et al., 1999; Allison et al., 1999; Henderson et al., 2005). In this regard the emerging pattern of variation in the molecular mechanisms of antipsychotic drugs in the face of their common clinical profiles resembles what was previously observed with the variability in neuroreceptor binding profiles (Kinon and Lieberman, 1996). The marked differences in the affinities and selectivity of the various antipsychotics for the receptors of different neurotransmitters were thought to underlie a rich pattern of clinical variation in the therapeutic actions of this group of drugs (Miyamoto et al., 2005). However, this hypothesis has not been supported by clinical studies (Lieberman, 2006; Lewis and Lieberman, 2008).

Nevertheless, there is reason to be hopeful that through functional selectivity, or other potential actions, the abilities of drugs to engage different signaling pathways will confer novel therapeutic effects that will improve the efficacy of treatments. In this context, the studies of Masri et al. (2008) and Klewe et al. (2008) highlight the plausibility that D2R/arrestin 3 modulation of Akt and GSK-3 activity is an important mechanism underlying psychosis and a potential target for future antipsychotic drugs. Further study of this pathway, including studies designed to reverse the effects of D2R antagonists or partial agonists (antipsychotic drugs) with systematic differential manipulation of the signaling pathways induced by D2R activation, is likely to be a fruitful path toward the development of novel treatments for schizophrenia-related disorders.

Acknowledgements: The authors would like to acknowledge the generous support of the Lieber Center for Schizophrenia Research at Columbia University